Medical researchers and clinical physicians often isolate and examine tissue, individual cells, and macromolecules to gain a greater understanding of their patient's needs. Macromolecules come in four basic types: proteins (structural molecules, enzymes, etc.), carbohydrates (sugars that are often involved in cell surface recognition), lipids (fats including those that make up cell membranes), and nucleic acids (DNA and RNA). Cells are made up of all of these macromolecules and a good deal of water and small ions (sodium, potassium, chloride, etc.) The isolation and purification of specific macromolecules from biological tissues is the traditional realm of biochemistry, though this treatment of nucleic acids is now considered the realm of molecular biology.
The localization of specific macromolecules in tissue sections can be achieved by a variety of forms of histochemistry. Histology is the study of tissues, usually by cutting thin sections of tissue and looking at it under a microscope, the source of illumination may be visible light (traditional light microscopy), ultraviolet light (fluorescent microscopy), or electrons (electron microscopy). If a chemical reagent is used to identify a specific substance the method is simply termed histochemistry, if a reaction involves enzymatic catalysis, the term is enzyme histochemistry. When specific antibodies are used to identify a particular antigen (or epitope) in a tissue section, the terms used are immunohistochemistry, immunocytochemistry, or immunolocalization. When specific complementary sequences of nucleic acids are used to hybridize with specific cellular nucleic acids in tissue sections, the term used is in situ hybridization histochemistry.
The macromolecules of animal tissues are typically extracted, isolated, and purified before being separated by gel electrophoresis. Once separated, these macromolecules are generally transferred and immobilized on a membrane before being probed with specific antibodies or nucleic acids in the powerful and popular techniques of Northern, Southern, and Western hybridization.
In contrast to conventional blotting, in which macromolecules are transferred to a membrane along with their relative electrophoretic positions, "tissue prints" are believed to transfer molecules (from cell juices) along with their relative tissue locations. Some components of the "cell juice" are transferred to membranes during the tissue printing of botanical tissues, however, there is no evidence yet that whole cells or groups of cells are transferred in this process.
The technique of transferring macromolecules (including proteins and nucleic acids) directly from intact tissues to nitrocellulose or other blotting membranes has been called "tissue printing" (Varner, 1992). This technique has recently gained popularity in the field of plant science in large part because of the work of Cassab and Varner (1987). Botanical tissues are particularly well-suited or tissue printing because of their characteristic structural rigidity and symmetrical tissue architecture. It is, however, possible to make tissue prints of animal tissues as well.
One step further than a tissue print is isolating individual intact cells on nitrocellulose. In Cell blotting: Techniques for staining and microscopical examination of cells blotted on nitrocellulose paper (Anal. Biochem. 157:331-342, 1986), Seshi taught that individual isolated cells--both normal and neoplastic cells--can be immobilized onto nitrocellulose membrane with retained cytological detail. Seshi fails, however, to realize that the cells need not be digested and isolated. All the architectural information is lost by digesting and suspending his cells. This paper discusses the use of immunological agents to detect specific molecules (in this case the Leu-4 antigen on lymphocytes and chromogranin). This study also employs specific cell adhesion molecules, in this case fibronectin, to enhance the binding of cells, in this case BHK cells, to fibronectin-treated nitrocellulose membrane during a 60 minute incubation.
In Ion channel expression by white matter glia: The type-i astrocyte (Barres BA, Koroshetz, Chun LLY, Corey P., Neuron 5:527-544, 1990.), the authors describe what they call, "a new `tissue print` dissociation procedure" to isolate individual cells from brain so that they could perform electrophysiology studies on them. These authors state that they wanted to use tissue printing to, "exploit this adhesion between tissues and membranes! for cell isolation" and that they developed "an new `tissue print` technique that produces dissociated cells . . . " (Barres et al., page 27). In some cases, brain tissue was partially digested before tissue printing onto nitrocellulose membrane; in other cases Vibrotome sections (50-100 micrometers thick) of brain tissue was printed onto nitrocellulose paper. These authors did recognize that live cells could be transferred to nitrocellulose substrata, but did not foresee the utility of the technique as a method of preserving the architectural arrangement of cells from large tissues, indeed, they developed the method for cell isolation so that they could perform electrical experiments on isolated cells with intact cell processes. In the discussion these authors state that, "The tissue print protocol is a simple variant of standard dissociation protocols." and that " . . . rather than shearing the tissue apart by passage through a syringe needle or pipette, a `touch prep` a technique used in clinical pathology! is prepared by gently touching the tissue to a sticky, nontoxic surface." They go on to say, "Unlike previous methods, this tissue print procedure allows isolation of viable cells still bearing processes for further study. Here we have used tissue prints for electrophysiological recording; however, we have also used the procedure for other purposes, including isolation of cells for culture, scanning electron microscopy, and immunohistochemistry." Notably, they do not mention transmission electron microscopy nor do they discuss anything about pathological tissues.
In Simultaneous recording of Ca2+!i increases in isolated olfactory receptor neurons retaining their original spatial relationship in intact tissue (Hirono J, Sato T, Tonoike M, Takebayashi M., J. Neurosci. Meth. 42:185-194, 1992) the authors teach that small slices of olfactory epithelium (i.e., smelling nerves) can be dissected and digested before "unrolling" a piece of tissue onto a glass substrate coated with the lectin Concanavalin A as an adhesive. These authors state that the "relative local arrangement between the cells" is preserved. It should be pointed out that this technique involves very small pieces of tissue with the purpose of preparing the cells for tissue culture and from the pictures shown, the tissue and organ architecture is poorly preserved.
Currently, there is no technique for transferring layers of animal tissue that retain the tissue and organ architecture and are viable when transferred.
The renal biopsy is an essential diagnostic tool for a wide variety of renal disorders. The procedure has, until recently, involved the use of 12-18 gauge needle devices, such as the vim-Silverman biopsy needle, which are associated with a small but significant risk of serious hemorrhage complications. Large gauge needles have fallen out of favor with the recent development and popularization of various biopsy gun devices, which are typically equipped with smaller (16-18 gauge) needles. These devices reduce patient morbidity, but also yield smaller biopsy specimens. As the assessment of glomerular pathology remains one of the most important objectives of the renal biopsy procedure, it is essential that each biopsy contain an adequate number of glomeruli. Whereas the mean yield from larger biopsy cores is routinely 25-75 glomeruli per core, the yield from smaller cores is often less than 10. At the lower end of this range the number of glomeruli is frequently inadequate to make a proper diagnosis.
It is possible to calculate the number of glomeruli within a cylindrical biopsy of known dimensions now that an unbiased estimate of the numerical density of glomeruli in normal autopsied human kidney has been reported. Such estimates are substantially higher than the number of glomeruli typically found in clinical renal biopsy specimens. This analysis, together with the clinical observation that spheroidal voids (the shape and size of a glomerulus) are observed on the biopsy surface, suggests that glomeruli become separated from the biopsy core during or after the biopsy procedure. Therefore, a need exists for improving the yield of the biopsy procedure.